Field of the Invention
The present invention discloses gene targets, constructs and methods for the genetic control of plant disease caused by plant-parasitic nematodes—specifically nematodes of the genus Pratylenchus known as root lesion nematodes. More specifically, the present invention relates to achieving plant protective effects through the identification of target coding sequences and the use of recombinant DNA technologies, to provide specific dsRNA sequences in the nematode diet, for post-transcriptionally repressing or inhibiting expression of the target coding sequences in the cells of plant-parasitic nematodes to provide plant protection. The gene targets disclosed in this invention are excellent candidates for generating robust protection against multiple Pratylenchus species via RNA interference methods. There is nonetheless sufficient nucleotide divergence to avoid cross reactivity with plant orthologs and to provide selectivity to other non-target organism such as mammals and beneficial insect species such as bee pollinators.
Description of Related Art
Plants are subject to multiple disease causing agents, including plant-parasitic nematodes, which are active, flexible, elongate organisms that live on moist surfaces or in liquid environments, including films of water within soil and moist tissues within other organisms. Nematodes grow through a series of lifecycle stages and molts. Typically, there are five stages and four molts: egg stage; J1 (i.e. first juvenile stage); M1 (i.e. first molt); J2 (second juvenile stage; sometimes hatch from egg); M2; J3; M3; J4; M4; A (adult). Juvenile (“J”) stages are also sometimes referred to as larval (“L”) stages.
Both plant-specific and animal-specific species of nematodes have evolved as very successful parasites and are responsible for significant economic losses in agriculture and livestock production and for morbidity and mortality in humans. Nematode parasites of plants can inhabit all parts of plants, including roots, developing flower buds, leaves, and stems. There are numerous plant-parasitic nematode species, including various lesion nematodes (i.e. Pratylenchus spp.), root knot nematodes (i.e. Meloidogyne spp.), cyst nematodes (i.e. Heterodera spp.), dagger nematodes (i.e. Xiphinema spp.) and stem and bulb nematodes (i.e. Ditylenchus spp.), among others. However, the largest and most economically important groups of plant-parasitic nematodes are the families Pratylenchidae (lesion nematodes), Meloidogynidae (root knot nematodes) and Heteroderidae (cyst nematodes) with lesion and root knot nematodes being particularly noteworthy for their very broad host rages. Plant parasitic nematodes are classified on the basis of their feeding habits into the broad categories of migratory ectoparasites, migratory endoparasites, and sedentary endoparasites. Sedentary endoparasites, which include the root knot nematodes (Meloidogyne spp.) and cyst nematodes (Globodera and Heterodera spp.) induce feeding sites (“giant cells” in the case of root knot nematodes and “syncytia” for cyst nematodes) and establish long-term infections within roots. In contrast, while spending most of their lifecycles within host tissues, migratory endoparasitic nematodes like lesion neamtodes (Pratylenchus spp.) do not induce permanent feeding sites but feed while migrating between or through plant cells. It is estimated that parasitic nematodes cost the horticulture and agriculture industries in excess of $78 billion worldwide a year, based on an estimated average 12% annual loss spread across all major crops. For example, it is estimated that nematodes annually cause soybean losses of approximately $3.2 billion worldwide (Barker et al., 1994).
Traditional approaches to control plant diseases have relied on crop rotation, chemical treatment and the construction of interspecific hybrids between resistant crops and their wild-type relatives as sources of resistant germplasm. However these traditional approaches all suffer from significant limitations for root lesion nematode control. For example, genetic resistance to lesion nematodes is usually narrow spectrum (e.g., resistance or tolerance to P. thornei in wheat is rarely accompanied by resistance to P. neglectus) whereas multiple species of lesion nematode are typically present in fields where crops are grown. In addition, most chemical nematode control agents are not effective in eradicating nematode infestations as nematodes inside roots are largely protected. The few agents like fumigant methyl bromide that can effectively get to nematode reservoirs are biocides effectively sterilizing a field for a period of time over which the nematode control agent has been applied. In addition, the most widely used fumigant nematicide, methyl bromide, is scheduled to be soon retired from use, and at present, there is no promising candidate to replace this treatment. The non-fumigant organophosphates and carbamates nematicides like ethoprop, terbufos, carbofuran and aldicarb though not as broad spectrum also show poor selectivity. In particular these chemical nematode control agents are highly toxic to mammals, birds, fish, and to non-target beneficial insects. These agents can in some cases accumulate in the water table or the food chain, and in higher trophic level species. These agents may also act as mutagens and/or carcinogens to cause irreversible and deleterious genetic modifications. As a result, government restrictions have been imposed on the use of these chemicals. Additionally, few chemical nematicides (fumigant or non-fumigant) are cost effective for use in large acreage row crops such as soybeans and corn. Chemical seed treatments can be economically applied in large acreage control crops but these only provide early season protection under moderate levels of nematode infestation. Finally, crop rotation or fallowing without weeding is not an effective strategy for controlling root lesion nematodes because of their broad host ranges which includes most crops, native grasses and weeds.
Consequently, alternative methods for nematode control, such as genetic methods, are increasingly being studied. An environmentally benign but effective alternative for controlling lesion nematodes is the use of RNA interference against essential nematode genes to control nematode infestation of plants. This is achieved through the transgenic expression of double stranded RNA (dsRNA) in plants complementary to target nematode genes. It was first demonstrated in the free living nematode Caenorhabditis elegans that dsRNA could be provided in the diet either through expression in bacterial food source or through soaking in dsRNA containing solutions and effect suppression of the genes function in the nematodes (Fire et al. 1998 Nature 391(6669):806-11, Fire et al. U.S. Pat. No. 6,506,559). RNA interference (“RNAi”) utilizes endogenous cellular pathways whereby a double stranded RNA (dsRNA) specific target gene results in the degradation of the mRNA of interest or diminished translation of protein from the mRNA template. The effector proteins of the RNAi pathway include the Dicer protein complex that generates ˜21-nucleotide small interfering RNAs (siRNAs) from the original dsRNA and the RNA-induced silencing complex (RISC) that uses siRNA guides to recognize and degrade or block translation from the corresponding mRNAs. Only transcripts complementary to the siRNAs are affected, and thus the knock-down of mRNA expression is usually sequence specific. In some systems the initial dsRNA trigger or siRNA effectors can be amplified by RNA Directed RNA Polymerases (RDRPs) in an mRNA template dependent manner which can lead to the production of secondary siRNAs both within and outside the initial trigger dsRNA. The gene silencing effect of RNAi can persist for days and, under experimental conditions, can in some cases lead to a decline in abundance of the targeted transcript of 90% or more, with consequent decline in levels of the corresponding protein. Protein levels can also be perturbed by blocking translation without significantly affecting mRNA transcript levels.
Recent reports suggest that such an approach may have potential for the control of plant parasitic nematodes (Steeves et al. 2006 Funct Plant Biol. 33(11): 991-999; Huang et al. 2006 Proc Natl Acad Sci USA. 103(39):14302-6; Yadav et al. 2006 Mol Biochem Parasitol. 148(2):219-22). However the selection of the gene target and the choice of promoter to drive the dsRNA are both crucial and not easy to predict a priori (Fairbairn et al. 2007 Planta 226(6):1525-33). Additionally to be most effective the dsRNAs must not cause yield drag due to phytotoxicity from unfavorable off target effects in the plant host. This invention discloses gene targets in Pratylenchus species which are suitable for providing genus wide, durable, commercial levels of nematode resistance through RNAi-based approaches without untoward effects to the host plant or non-target organisms.